1. Field of the Invention
This invention relates to a method for integrating a gene fragment inserted between a pair of transposon sequences into a chromosome of a mammalian cell, comprising introducing at least one expression vector which comprises a gene fragment comprising a DNA encoding a protein of interest and also comprises a pair of transposon sequences at both terminals of the gene fragment, into a suspension mammalian cell; and a method for producing the protein comprising suspension-culturing a suspension mammalian cell which produces the protein, a suspension mammalian cell which expresses the protein; and an expression vector which comprises a gene fragment comprising a DNA encoding a protein of interest and also comprises a pair of transposon sequences at both terminals of the gene fragment.
2. Brief Description of the Background Art
Production of exogenous proteins by recombinant DNA techniques is used in various industries such as pharmaceutical industry and food industry. In most cases, production of recombinant proteins is carried out by introducing an expression vector comprising a nucleotide sequence encoding a protein of interest into a host, such as Escherichia coli, yeast, insect cell, plant cell, and animal cell, selecting a transformant in which the expression vector is integrated into the chromosome, and further culturing the transformed cell line under appropriate culture conditions.
However, in order to develop a host which can produce an exogenous protein efficiently, it is necessary to select a host cell having good productivity for each protein of interest, so that a further technical innovation is desired on the exogenous protein production techniques for each host.
In the bacteria systems, such as Escherichia coli, and yeast systems, different from animal cells, post-translational modifications, such as sugar chain modification, are difficult to attain in many cases and thus cause a problem in producing a protein having its activity.
Since the produced protein is subject to a post-translational modification such as phosphorylation and addition of sugar chains in the insect system, this system has a merit that the protein having its original physiological activity can be expressed. However, since the sugar chain structure of the secreted protein is different from that of mammalians-derived cells, antigenicity and the like become a problem when the protein is applied to pharmaceutical use.
In addition, since a recombinant virus is used in the insect cell system when an exogenous gene is introduced, there is a problem that its inactivation and containment of the virus are required from the viewpoint of safety.
In the animal cell system, post-translational modifications, such as phosphorylation, sugar chain addition, and folding, can be conducted to proteins derived from higher animals including human, in more similar manner to those produced in the living body. Such accurate post-translational modifications are necessary for recreating the physiological activity originally possessed by a protein in its recombinant protein, and a protein production system in which a mammalian cell is used as a host is usually applied to pharmaceutical products and the like that requires such physiological activity.
However, a protein expression system in which a mammalian cell is used as the host is generally low in productivity, and also causes a problem of the stability of introduced genes in many cases. Improvement of productivity of a protein using a mammalian culture cell as a host is not only very important in producing medicaments for treatment, diagnostic agents and the like, but also greatly contributes to research and development of them. Thus, it is urgent to develop a gene expression system which easily makes it possible to obtain a cell line of a high productivity using a mammalian culture cell, particularly Chinese hamster ovary cell (CHO cell), as the host.
A transposon is a transposable genetic element which can move from one locus to other locus on the chromosome. A transposon is a strong tool for the study on molecular biology and genetics and used for a purpose, such as mutagenesis, gene trapping, and preparation of transgenic individuals, in insects or nematode (e.g., Drosophila melanogaster or Caenorhabditis elegans) and plants. However, development of such a technique has been delayed for vertebral animals including mammalian cells.
In recent years, however, transposons which have activities also in vertebral animals have been reported, and some of them were shown to have an activity in mammalian cells, such as cell derived from mouse and human. Typical examples include transposons Tol1 (Patent Reference 1) and Tol2 (Non-patent Reference 1) which are cloned from a medaka (killifish), Sleeping Beauty reconstructed from a non-autonomous transposon existed in Onchorhynchus fish genome (Non-patent Reference 2), an artificial transposon Frog prince (Non-patent Reference 3) which is derived from frog and a transposon piggyBac (Non-patent Reference 4) which is derived from insect.
These DNA transposons have been used for mutagenesis, gene trapping, preparation of transgenic individuals, expression of drug-resistant proteins, and the like, as a gene introduction tool for bringing a new phenotype in a genome of a mammalian cell (Non-patent References 5 to 12).
In the case of insects, a method in which an exogenous gene is introduced into silkworm chromosome using the transposon piggyBac derived from a Lepidoptera insect to express the protein encoded by said exogenous gene has been studied, and a protein production method using the above techniques was disclosed (Patent Reference 2).
However, since protein of interest is not expressed at sufficient levels and is produced in the whole body of silkworm, it causes an economical problem due to the necessity of an advanced purification technique for recovering the expressed exogenous protein in a highly purified form from the body fluid including a large amount of contaminated proteins.
In addition, an example in which a protein relating to G418 resistance is expressed in a mammalian cell using the medaka-derived transposon Tol2 (Non-patent References 12 and 13) is known.
In the case of producing a protein drug for medical use using a mammal-derived cultured cell, it is important that an animal-derived component is not contained during its production process in order to prevent unexpected contamination of an unknown virus or pathogenic polypeptide. CHO cell is most frequently used as an animal cell for producing a protein drug, and due to the studies of recent years, a suspension CHO cell line capable of culturing in a safe medium which does not use a serum or animal-derived component has also be established. However, productivity of a cell line into which a gene was introduced under a serum-free or protein-free condition is limited to half that of the cell line into which a gene was introduced under a serum-used condition (Non Patent Literature 14). It is shown that gene transduction under a serum-free or protein-free condition is technically difficult.
It is general that a selectable marker for screening a cell expressing a protein of interest is arranged on the same gene expression vector. This is based on a hypothesis that there are a region where a gene existing in the genome is easily expressed and a region where a gene existing in the genome is hardly expressed (called as position effects, Non Patent Literature 15), and that the protein of interest is also expressed when the selectable marker is expressed.
On the other hand, when a protein of interest, is comprised of two or more polypeptides such as an antibody and the like, it is also known that each polypeptide is expressed using different vectors. In the case of an antibody, it has been shown that the productivity is higher when expression of heavy chain of the antibody is higher than the expression of light chain (Non Patent Literature 16). Since it is predicted that expressions of heavy chain and light chain become constant on the same vector. It becomes possible to obtain a cell line which expresses the heavy chain and light chain at an optimum ratio by intentionally expressing the heavy chain and light chain using different vectors for the purpose of obtaining high productivity. However, when a protein is expressed using two or more different vectors, two or more selectable marker genes are also necessary.
As a way for overcoming this, it was reported a case in which a dhfr gene originally consisting of one polypeptide chain was divided into two polypeptide chains and one of them was arranged on a heavy chain expression vector, and the other was arranged on a light chain expression vector (Non Patent Literature 17).
However, the cell described in the Non Patent Literature 17 is a CHO cell in which the cell is dependent on the protein component added to the medium, and as described in the above, there is a possibility that the gene introduction efficiency is high different from the case of the gene introduction under a serum-free or protein-free condition. It is predicted that selecting a cell of high productivity is still difficult when a gene is introduced under a serum-free or protein-free condition having high safety and free from the danger of viral infection and the like.